Our major objective in this proposal is to use insertional mutagenesis to identify, isolate, and characterize genes which play critical roles in mammalian embryogenesis. Studies of mammalian embryogenesis have been impeded by the lack of developmental mutations that can be molecularly defined, and thus associated with a cloned gene and protein product. Recently it has become evident that the transgenic mouse system provides a powerful experimental method for the generation of developmental mutation that can be studied at both the molecular and phenotypic levels. These mutations are produced by the random insertion of exogenous DNA into the mouse genome and the consequent inactivation of developmentally essential genes. A unique feature of such mutations is that the exogenous DNA serves both as a mutagen and as a molecular probe for cloning the mutant locus. We have identified a recessive insertional mutation in a transgenic mouse line which arrests development at the peri-implantation stage. The site of insertion on chromosome 3 has been designated the Rbeta3 locus. Both the wild type and mutant loci have been cloned and transcripts encoded by this locus have been detected in liver and kidney, as well as a number of other tissues. In project I, cDNA cloning and nucleotide sequencing experiments will be performed to characterize the developmentally essential gene at this locus, and to determine the identity of the encoded protein. Gene rescue experiments will be carried out in transgenic mice to confirm that the inactivation of this gene is responsible for the embryonic lethal phenotype. The cell type distribution of the mRNA and protein products of the Rbeta3 gene will be established by in situ hybridization and immunohistochemistry to lay the groundwork for future investigations of the function of this gene in the implanting embryo. In project II, we propose to generate new insertional mutations by the microinjection of mouse eggs using a construct, CAT-IN.1, designed specifically to facilitate the identification and molecular cloning of recessive insertional mutations. 100-200 transgenic lines carrying CAT-IN.1 will be produced and screened for recessive mutations by a quantitative CAT assay. The stage of developmental arrest and the chromosomal location of each insertional mutation will be established. These mutations promise to provide new molecular probes for key stages in mammalian development.